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Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb

Adsorption properties of biologically active derivatives of quaternary ammonium surfactants and their mixtures at aqueous/air interface II. Dynamics of adsorption, micelles dissociation and cytotoxicity of QDLS Monika Rojewska a , Krystyna Prochaska a,∗ , Anna Olejnik b , Joanna Rychlik b a b

Poznan University of Technology, Institute of Chemical Technology and Engineering, Pl. M. Skłodowskiej-Curie 2, 60-965 Pozna´ n, Poland Department of Biotechnology, Food Microbiology, Poznan University of Life Sciences, ul. Wojska Polskiego 48, 60-627 Pozna´ n, Poland

a r t i c l e

i n f o

Article history: Received 30 January 2014 Received in revised form 14 March 2014 Accepted 19 March 2014 Available online xxx Keywords: Catanionic mixtures Adsorption dynamics Micelle dissociation rate constant Diffusion coefficient In vitro cytotoxicity

a b s t r a c t The main aim of our study was analysis of adsorption dynamics of mixtures containing quaternary derivatives of lysosomotropic substance (QDLS). Two types of equimolar mixtures were considered: the ones containing two derivatives of lysosomotropic substances (DMALM-12 and DMGM-12) as well as the catanionic mixtures i.e. the systems containing QDLS and DBSNa. Dynamic surface tension measurements of surfactant mixtures were made. The results suggested that the diffusivity of the mixed system could be treated as the average value of rates of diffusion of individual components, micelles and ion pairs, which are present in the mixtures studied. Moreover, an attempt was made to explain the influence of the presence of micelles in the mixtures on their adsorption dynamics. The compounds examined show interesting biological properties which can be useful, especially for drug delivery in medical treatment. In vitro cytotoxic activities of the mixtures studied towards human cancer cells were evaluated. Most of the mixtures showed a high antiproliferative potential, especially the ones containing DMALM-12. Each cancer cell line used demonstrated different sensitivity to the same dose of the mixtures tested. © 2014 Elsevier B.V. All rights reserved.

1. Introduction The substances exhibiting a strong surface activity towards membranes of organelles play a key role in many biological processes such as in bone resorption, tumour cell progression and programmed processes of cell death. In this paper we present results of a study of cationic surfactants from the group of quaternary derivatives of lysosomotropic substances (QDLS). Quaternary derivatives of lysosomotropic substances exhibit both surface activity as well as fungicidal and bactericidal activities and show antistatic and hemolytic properties [1]. However, the most interesting is the possibility of these substances application in medicine [2–5]. Lysosomotropic compounds show special biological properties [6–8]. However, up to now the mechanism of activity of lysosomotropic substances has not been fully recognised, and therefore it is worthwhile to delve deeper into the surface properties of these compounds. Especially interesting seem to be the

∗ Corresponding author. Tel.: +48 616653601; fax: +48 616653649. E-mail address: [email protected] (K. Prochaska).

binary surfactant systems of QDLS and catanionic mixtures (i.e. systems containing two ionic surfactants of the opposite charge) because these mixtures usually show better adsorption properties than individual compounds [9–11]. As a result, mixtures of surfactants show lower surface tension and/or lower value of CMC than the single compounds at the same concentration. Surfactant mixtures have been studied extensively over the years and literature gives much information about the formation of mixed adsorption monolayers and micelles in them. A lot of papers have reported on the synergistic effects in adsorption properties of surfactant mixtures [12–16]. Synergistic behaviour has also been observed in biological activity of some cationic–anionic mixtures of surfactants [9,11]. Many interfacial phenomena are caused by the non-equilibrium of the adsorption layer. Hence, adsorption dynamics of a surfactant solution plays an important role in determining the system behaviour at interface. Dynamics of adsorption in mixed surfactant solutions has been widely discussed in literature [17–20]. Frese et al. [21] have presented the effect of dissociation kinetics of mixed micelles on the adsorption dynamics of the catanionic mixture. The dynamics of surface tension of micellar surfactant

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solutions significantly depends on the diffusion rates of monomers and micelles and the dissociation/dissolution process of the micelles. Aggregates are additional sources of surfactant molecules which are transported as monomers to the interface. Analysis of adsorption dynamics of the mixtures tested should be made taking into account the time of aggregates formation and duration of the diffusion process of monomers, micelles and ion pairs at the interface. Investigation of adsorption dynamics is also important from the perspective of biomedical applications of QDLS and their mixtures. The catanionic mixtures have been proposed to improve the delivery of drug compounds and DNA or drugs for HIV treatment [22–24]. Moreover, mixed micelles are potential vehicles for the controlled release of drugs over time [25,26] or other compounds as needed in for particular tasks in the pharmaceutical, pesticide, food and cosmetics industries. The release of the vesicle core contents requires that the contents cross the bilayer shell. If the contents are surface active, this transport may include the dynamics of adsorption into one leaflet of the bilayer, a subsequent rearrangement, and desorption at the other side [27]. In this paper we present results concerning adsorption dynamics as well as micellar properties of mixed surfactant systems containing QDLS and exhibiting specific biological properties [9–11,28]. In our previous paper [11] we analysed surface activity of mixtures containing QDLS derivatives and in some mixed systems we found synergistic effects in reduction of both surface tension and CMC values. The present work was undertaken with the primary aim of studying the dynamics of adsorption process and aggregation behaviour in binary mixtures containing two QDLS derivatives, as well as in catanionic mixtures containing QDLS and sodium dodecylobenzenesulphonate (DBSNa) as an anionic surfactant. The research work presented was stimulated by the fact that mixed systems are used in many pharmaceutical formulations [29,30]. It is probable that in the presence of a catanionic system, the anionic compound, mainly alkylsulphonate, gets incorporated in a lipid bilayer, modifies the electrostatic barrier and thus facilitates the transport of the cationic component [26,31]. In order to better understand the mechanism of transport of lysosomotropic substances across cell membranes, the incorporation phenomena of catanionic mixture into phospholipid monolayer (dipalmitoylphosphatidylcholine, DPPC) are planned to be investigated in the next stage of our studies. In our previous paper [11] we have shown that catanionic mixtures containing QDLS can suppress the growth of cancer cells Caco-2 and reduce their cell viability in a dose-dependent manner. In this paper we expand the scope of biological research over other human cancer cell lines.

tension measurements were performed using the bubble pressure tensiometer (Sita t60), which recorded changes in surface tension in times from 30 ms to 60 000 ms. On the basis of the dynamic surface tension data for a micellar solution the magnitude of micelle dissociation rate constant was estimated from the equation [21,36]:



[d/dt 1/2 ]CMC k2  =˛ 4 [d/dt −1 ]c>CMC

1/2

  1/2

=˛

4

 −1/2

(1)

Here k2 is the micelle dissociation rate constant which is inversely proportional to the relaxation time of the slow micelle kinetic process , ˛ is the relative concentration of monomers at c > CMC. For ionic surfactants it can be assumed that ˛ = 1 up to a concentration 10 mM. The derivative d/dt−1/2 was determined from the plot  (t−1/2 ) for t → ∞ at c = CMC and the derivative d/dt−1 was determined from the plot  (t−1 ) for t → ∞ at a concentration above CMC. The surface activity of a mixture depends on the composition of the mixed micelles formed. Therefore, in literature several models have been proposed for the description of the structure of mixed micelle composition [12,15,37–39]. In this paper, the values of mole surface fractions (X), interaction parameters between molecules in the micelles (␤M ) and free energy of micellization (Gm ) were estimated on the basis of the Rubingh model [12]. According to the Rubingh model, the relevant equations are: ˇM =

ln(˛CMC12 /X1M CMC1 ) (1 − X1M )

(2)

2

where ˛ is the mole fraction of surfactant 1 in the mixture of two surfactants, X1M is the mole fraction of surfactant 1 in mixed micelles, respectively. CMC1 and CMC12 are the CMCs values of individual surfactants and of their mixture at a certain value of ˛. As shown by Rosen, the two conditions for a synergism in formation of mixed micelles can be expressed by Eq. (3):

 M   CMC1  ˇ  > ln  CMC

ˇM < 0;

(3)

2

A quantitative description of adsorption kinetics processes is usually based on the model derived in 1946 by Ward and Tordai [40]. It is based on the assumption that the time dependence of surface/interfacial tension, which is directly related to the surface/interfacial concentration  of adsorbed molecules via the equation of state, is mainly caused by the surfactant transport to the interface.



 (t) = 2



D ˘

√ c0 t −



√

t

√ c(0, t − ) t



(4)

0

2. Experimental and theoretical methods Two derivatives of lysosomotropic substances obtained by quaternisation of the appropriate tertiary amino compounds with methyl bromide: alkyl N,N-dimethylalaninates methobromides (DMALM-12) and alkyl N,N-dimethylglycinates methobromides (DMGM-12) were studied. The synthesis and biological properties of the components of mixtures investigated are described in details in [32–35]. The chemical structures and adsorption properties of the compounds analysed are presented in [11]. Two types of surfactant mixtures were investigated: a mixture of two cationic compounds: DMALM-12 + DMGM-12 at different molar ratios and catanionic mixtures consisting of DMALM-12 or DMGM-12 as the cationic QDLS and sodium dodecylbenzenesulphonate (DBSNa) as the anionic component. The water used as solvent for preparing all aqueous solutions was deionized and purified to a final resistivity 18.2 M cm by a PURELAB Classic UV system (ELGA LabWater). Dynamic surface

where D is the diffusion coefficient and c0 is the surfactant bulk concentration. The integral equation describes the change in  (t) with time t. The application of the Ward and Tordai Eq. (4) to dynamic surface tension data (t) is not simple and is often avoided because of numerical difficulties. Various models developed on this basis differ mainly in the use of different boundary and initial conditions [41]. The simplest and often used short time approximation is:

  = 2c0

Dt ˘

(5)

obtained from Eq. (4) by neglecting the integral. Using the linear relation between  and  the interfacial tension of a surfactant solution at t → 0 is given then by:



t→0 = 0 − 2nRTc0

Dt ˘

(6)

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where  0 is the surface tension of the solvent and n is 1 for nonionic and 2 for ionic√surfactants, respectively. The derivative of Eq. (6) with respect to t yields:



 d  √ d t

= −2nRTc0 t→0

Dt ˘

(7)

where D is the diffusion coefficient for short times of adsorption, c0 is the surfactant bulk concentration, ˘ is the surface pressure, R is temperature and the slope of dynamic the gas law constant, T is the √ surface tension isotherm ( t) is equal to the expression on the right hand side of Eq. (7). The experimental values in the form of √ dynamic surface tension isotherm ( t) must give a straight line [42]. The effect of the surfactant mixtures containing QDLS on cell viability was determined by in vitro cytotoxicity assay. Human cancer cells HT-29 (colon carcinoma), HepG2 (liver cancer) and MCF-7 (breast cancer) were obtained from The European Collection of Cell Cultures (ECACC, UK) supplied by Sigma–Aldrich. The cells were cultured in Dulbecco’s Modified Eagle Medium (Sigma–Aldrich) supplemented with 10% foetal bovine serum (Gibco® ), 1% nonessential amino acids (Sigma–Aldrich) and 50 mg L−1 gentamycin (Gibco® ). The nontransformed, nontumorigenic colon NCM460 cell line, derived from human normal colon mucosa [43], was provided by INCELL Corp. LLC (San Antonio, USA) and was grown in M3 Base medium (INCELL Corp. LLC) with 10% foetal bovine serum addition. The cells were seeded at initial density of 2.5 × 104 cells/cm2 in 96-well plates and maintained at 37 ◦ C in a humidified, 5% CO2 , 95% air atmosphere. At twenty four hours after seeding, the cells were exposed to the DMALM-12, DMGM-12, DBSNa derivatives and their equimolar mixtures at concentrations ranging from 3.8 to 125.0 ␮mol L−1 and incubated for 48 h. The effect of derivatives tested on cellular viability was evaluated using an assay based on the cleavage of the yellow dye MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Sigma–Aldrich) to purple formazan crystals by dehydrogenase enzymes in mitochondria, which is a conversion that occurs only in metabolically active cells [44]. In the present study, the MTT assay was performed according to the modified method described by Vian, Vincent, Maurin, Fabre, Giroux and Cano [45]. Briefly, after treatments, to each well a medium containing 0.5 mg MTT/ml was added, and the microplate was incubated at 37 ◦ C for 3 h. At the end of the incubation period, acidic isopropanol was added to each well. The plate was shaken for 20 min to dissolve formazan. The absorbance was measured at 570 nm using a TECAN M200 Infinite microplate reader. The cell viability was expressed as a percentage of absorbance compared to that of the control (untreated cells). Experimentally derived data are shown as mean ± SD (n = 3). 3. Results and discussion 3.1. Dynamics of adsorption of surfactant mixtures at the air/water interface Dynamics of adsorption of mixed systems is strongly dependent on surfactant concentrations and the presence of micelles in solution. As mentioned above, the stability of aggregates in the micellar solution has a significant influence on its surface activity. It is known that relatively unstable micelles support the adsorption process supplying additional monomers at the surface, while stable micelles cannot provide monomers fast enough [21,22]. For this reason the stability of micelles has a significant effect on the rate of surface tension reduction and hence on the course of the dynamic surface tension curve. A measure of the aggregates stability is the approximate value of the micelle dissociation rate constant (k2 ) in a given solution. As mentioned above, the dynamic surface tension

3

data were also used to calculate the micelle dissociation rate constants (k2 ). The values of k2 for individual components and their mixtures calculated according to Eq. (1) are presented in Table 1. The results obtained (Table 1) indicate a strong correlation between the surfactants concentration and the micelle dissociation rate constant in solutions studied. An increase in the surfactant concentration leads to formation of less stable aggregates in solutions of individual components as well as in their mixtures (increase in k2 ), regardless of molar composition of the investigated mixtures. Analysis of the value of k2 obtained for solutions of individual components has shown that the cationic surfactants form less stable micelles than the anionic surfactant DBSNa. The k2 values estimated for QDLS solutions of concentrations close to 2 CMC were used to compare the stabilities of micelles formed in micellar QDLS solutions. It is clearly evident that the least stable micelles are formed in a solution of DMALM-12. In the catanionic system DMGM-12 + DMALM-12, the most stable micelles are formed in the equimolar mixture as evidenced by the value of k2 is by one order of magnitude lower than for nonequimolar ones. The estimated values of k2 are comparable with those obtained for DMALM-12 + DMGM-12. Moreover, the mixed micelles are much more stable than the aggregates formed in solutions containing only individual QDLS. Fig. 1a and b presents the curves of dynamic surface tension for DMGM-12 + DMALM-12 mixture at different molar ratios, both in a non-micellar and micellar solution. In the micellar solutions of the mixtures studied, the synergistic effect in lowering the surface tension was not observed, however it was the lowest for the equimolar mixture. Analysis of the values of diffusion coefficients (Dt→0 ) for the composition of DMGM-12 + DMLAM-12 at different molar ratios (Table 1) indicates that the diffusion coefficient estimated for equimolar mixture is approximately 24 × 10−12 m2 /s. It is worth stressing that the value of diffusion coefficient obtained for the equimolar system is almost tenfold the values of Dt→0 for non-equimolar mixed compositions (1:3 and 3:1). As a result, one can expect that the particles are able to reach the interface in shorter time and faster reduce the surface tension of the equimolar solution. In the equimolar mixture in non-micellar solutions the synergistic effect in lowering the surface tension was observed. As shown in our previous paper [11] all catanionic mixtures investigated show synergistic effect in lowering the CMC because the values of CMC of catanionic mixtures are significantly smaller than those expected from the weighted average, determined on the basis of CMC of each components and the parameter ˛ as a weighting factor with respect to Eq. (3). In addition, it is worth noting that the shapes of dynamic surface tension curves for the equimolar mixture of DMGM-12 + DMALM-12, both in non- and micellar solutions, correspond to the character of curves of dynamic adsorption obtained for DMGM-12. On the basis of dynamic surface tension isotherms it can be assumed that for DMGM-12 + DMALM-12 mixture in the mixed adsorption monolayer formed at the air/water interface the content of DMGM12 derivative can dominate. This effect may be due to the higher diffusivity of the DMGM-12 derivative than that of DMALM-12, as evidenced by the values of diffusion coefficients presented in Table 1. The synergistic effect of lowering the surface tension in micellar solutions of catanionic mixtures is illustrated in Fig. 2b and d. This effect was observed after the adsorption time longer than 10,000 ms. Thus, it can be concluded that the electrostatic interactions in the mixed cationic–anionic system considered not only cause a decrease in CMC and an increase in the efficiency of adsorption [11], but also accelerate the dynamics of adsorption. Dynamic surface tension curves for catanionic mixtures at different molar concentrations are presented in Fig. 2a and c. A rapid decline in the surface tension was observed with increasing concentration of the mixtures. The effect observed may be related to the micelles

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Table 1 Dynamic adsorption parameters: ␣1 – molar fraction of cationic surfactant in binary solution, k2 – micelles dissociation rate constant at 21 ◦ C, Dt→0 diffusion coefficient. ␣1

Component/mixture DMGM-12

N

+

k2 [s−1 ]

Dt→0 × 1012 [m2 /s]

8.158

1.707

5.0

0.143 (2 CMC)

2.316

0.1 10

– 1.200 (2 CMC)

6.592 1.713

–

7.611

10 –

CH3 H3 C

c [mmol L−1 ]

CH2COOC12H25 Br

-

CH3 DMALM-12

CH 3 N

H3C

+

–

CH 3 CHCOOC 12 H25 Br

-

0.1

CH 3 DBSNa

10

CH3(CH 2)10 CH2

S

15.68

3.774

–

O O Na

6.0

0.066

6.853

O

0.25 DMGM-121 + DMALM-12

0.50 0.75

2.0

0.027 (2 CMC)

10 6.0 4.0 1.0 4.0 10 4.0 2.0

2.404 0.389 (2 CMC) 0.097 – 0.069 (2 CMC) 34.90 0.262 (2 CMC) 0.105

2.942 7.459 9.171 12.95 24.07 2.519 11.39 14.23

42.22

DMGM-121 + DBSNa

0.50

10 1.0 0.4

3.768 0.256 0.031 (2 CMC)

0.147 0.792 10.88

DMALM-121 + DBSNa

0.50

10 1.0 0.4

8.298 0.831 0.077 (2 CMC)

0.414 4.127 8.717

stability (k2 ). For catanionic mixtures in concentration of 10 mM, the values of k2 are higher in comparison to those obtained for the series of diluted solutions of mixtures. The faster dissociation of micelles at 10 mM concentration causes a quick release of monomers, which reach the interface and reduce the surface tension in a really short time. The value of k2 for DMALM-12 + DBSNa mixture is higher than that of DMGM-12 + DBSNa mixture. As a consequence, in the system with

DMALM–12 + DBSNa, a significant lowering of surface tension to 60 mN/m within just 30 ms was observed. On the other hand, a characteristic plateau is found in the curves of adsorption dynamics of catanionic mixtures. The occurrence of this characteristic plateau is explained by the difference in the surface activity of the adsorbing components. This effect is particularly pronounced for the mixture consisting of a faster, less surface–active surfactants, and slower, but more active derivatives

Fig. 1. Dynamic surface tension isotherms for: (䊉) DMGM-12, () DMALM-12 and DMGM-12 + DMALM-12 mixtures at different molar ratio of components: () 1:3, () 1:1, () 3:1; (a) micellar solution c = 4 mM; (b) premicellar solution c = 1 mM; (c) effect of temperature on dynamics of adsorption for equimolar mixtures DMGM-12 + DMALM-12 at different concentration: (, ) c = 4 mM, (䊉, ) c = 2 mM, (, ) c = 1 mM, (, ) c = 0.1 mM, 21 ◦ C (black points) and 36.6 ◦ C (grey points).

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Fig. 2. Dynamic surface tension isotherms for: (a) DMGM-12 + DBSNa mixture: ( ) c = 10 mM, ( ) c = 1 mM, ( ) c = 0.4 mM, ( ) c = 0.1 mM, ( ) c = 0.04 mM, ( ) c = 0.01 mM, ( ) c = 0.001 mM; (b) micellar solution c = 10 mM of ( ) DMGM-12 + DBSNa mixture, (䊉) DMGM-12 and ( ) DBSNa; (c) DMALM-12 + DBSNa mixture: ( ) c = 10 mM, ( ) c = 1 mM, ( c = 0.4 mM, ( ) c = 0.1 mM, ( DMALM-12 and ( ) DBSNa.

) c = 0.04 mM, (

) c = 0.01 mM, (

) c = 0.001 mM; (d) micellar solution c = 10 mM of (

[46]. Hence, the compound, which later reaches the interface, wants to replace a less active compound but it needs much time to get to the interface. Jiang et al. [47] have shown that the isotherm plateau is related to the replacement of monomers rapidly diffusing to the interface by slower moving and more active complexes of the oppositely charged surfactants. It should be noted that the longer “arm” of the dynamic isotherms is observed for premicellar solutions. In a solution of a lower concentration the chance of forming ion pair is smaller, and therefore, rapidly diffusing monomers desorbed at the surface could be replaced by more active ion-pairs however at longer times. Moreover, ion-pair complex formed may need more time to get adsorbed and get suitably arranged at the interface. These results could be reflected in the shape of the adsorption curves as the longer “arm” of the isotherm (Table 2). The estimated values of diffusion coefficients for the micellar solutions of catanionic mixtures (at 2 CMC) are similar and are obtained as the mean value of the diffusion coefficients of individual components. Thus, these results seem to suggest that the diffusivity of the mixed system could be considered as the average of the rates of diffusion of individual components, micelles and ion pairs found in mixtures. Eastoe et al. [48,49] have shown that the adsorption of surfactant mixtures is diffusion controlled but only when the adsorption process in short time is considered. Therefore, we decided to analyse only the values of diffusion coefficients

) DMALM-12 + DBSNa mixture, ()

estimated for adsorption process which takes place in a short time. Analysis of the adsorption process in a long time requires taking into account other phenomena that contribute to the appearance of adsorption barrier, besides the diffusion process. For non-micellar mixtures the diffusivity was greater than for micellar mixtures, probably due to the fact that transport of single molecules to surface is easier than micelles or ion-pairs which are formed in micellar mixtures. Individual components of the mixture studied exhibit biological activity hence it is important to carry out tests in temperatures similar to that of the cellular environment. On the basis of the experimental curves of dynamic surface tension (Fig. 1c), it can be concluded that with increasing temperature the dynamic surface tension is decreased both in non- and micellar solutions of DMALM-12 + DMGM-12, but this effect does not change the shape of the curves. Table 1 shows the values of the micelle dissociation rate constant (k2 ) estimated at two temperatures: 25 ◦ C and 36.6 ◦ C. The value of k2 is by one order of magnitude higher at 36◦ than at 25 ◦ C. Thus, as expected, the increase in temperature causes a decrease in the stability of the micelles existing in the solution. 3.2. Cytotoxicity of surfactant mixtures Surface-active agents are widely used in development of new pharmaceutical dosage forms to improve the bioavailability of

Table 2 Synergistic effect in catanionic mixtures: ␣1 – molar fraction of cationic surfactant in binary solution, CMC – critical micellar concentration, Gm – free energy of micellization, XM – is mole fraction of DMGM-12 in mixed micelles, ␤M – molecular interaction parameters for the micelles (Eq. (2)). Component/mixture

␣1

CMC [mmol L−1 ]

−Gm [kJ mol−1 ]

XM

Reduction of CMC ␤M

DMGM-12 DMALM-12 DBSNa

– – –

2.3 4.6 0.924

43.4 40.4 48.4

– – –

DMGM121 + DMALM12 DMGM-121 + DBSNa DMALM-121 + DBSNa

0.25 0.50 0.75 0.50 0.50

2.80 1.92 1.66 0.035 0.038

43.5 45.2 45.8 68.8 62.5

0.435 0.498 0.533 0.473 0.455

– – – −11.3 −12.3 −13.4 −14.87 −15.7

 CMC  ln CMC1  2

– – –

0.072 0.911 1.605

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Table 3 IC50 for equimolar mixtures and their components. Compounds and mixtures

DMGM-12 DMALM-12 DBSNa DMALM-12 + DMGM-12 DMGM-12 + DBSNa DMALM-12 + DBSNa

IC50 (␮mol L−1 ) HepG2 cells

MCF-7 cells

HT-29 cells

78.30 ± 7.09 5.54 ± 1.00 >125 12.92 ± 2.68

112.08 ± 4.58 9.34 ± 1.41 >125 13.82 ± 1.54

79.53 ± 4.24 3.46 ± 0.27 >125 4.29 ± 0.83

107.50 ± 1.87 11.97 ± 4.40

drugs which reveal low solubility in water. They can influence the drug permeability by modifying barriers [50], by micellar solubilization, membrane fluidization and ion-pair formation. On the other hand, the activity of surfactants can lead to membrane damage and cell death and, therefore, in vitro tests are required for the investigation of cytotoxicity and irritative effects. In our experiments, the cytotoxicities of two types of surfactants mixtures and their components were evaluated. In our previous paper [11] we found a synergistic effect in reduction of proliferation of Caco2 cell. Now, we wanted to expand the study of cytotoxicity and simultaneously get more information on the biological activity of the mixtures tested. For this purpose, the cytotoxic potential of the mixture containing QDLS and their components were determined against human cancer cells derived from colon (HT-29 cell line), liver (HepG2 cell line), breast (MCF-7 cell line) and in nontransformed, nontumorigenic cells isolated from human normal colon mucosa (NCM460 cell line). Table 3 presents the values of IC50 for QDLS and their equimolar mixtures. DMALM-12 exhibits a high biological activity towards human cancer cell lines (HepG2, MCF-7, HT-29, Caco-2 [11]). DMALM-12 in the concentration range from 3 to 10 ␮mol L−1 causes a 50% reduction in viability and metabolic activity of the cancer cells (IC50 , Table 3). DMALM-12 showed the highest cytotoxicity towards HT-29 cells, and then HepG2, MCF-7, respectively. Addition of DMALM-12 both to DBSNa or DMGM-12 (at equimolar ratio of concentrations) results in a disproportionate increase in the cytotoxic activity of DBSNa and

63.54 ± 12.86 17.18 ± 3.91

94.28 ± 6.44 4.57 ± 0.38

DMGM-12 towards all cancer cell lines studied. Moreover, the strength of the toxic effect is much higher than that expected from the weighted average of IC50 values obtained for individual components of the mixed system. On the other hand, DMALM-12 also shows a strong cytotoxic effect towards untransformed NCM460 cells. The values of DMALM-12 cytotoxic doses (IC50 ) evaluated in normal (NCM460) and cancer (HT-29) cells derived from the colon epithelium are comparable, and the differences are statistically insignificant. The results indicated no selective cytotoxic effects of DMALM-12 towards specific cancer cells. Comparison of IC50 values (Table 3) indicates that some cell lines are markedly less sensitive to the cytotoxic effects of the surfactants. Analysis of the biological effects of cationic surfactants on the cancer cell lines shows that DMGM-12 has a significantly lower cytotoxic activity than DMALM-12. The colon cancer Caco-2 cells (IC50 = 39.7 ␮mol L−1 ) showed the greatest sensitivity towards DMGM-12 [11], whereas the breast cancer MCF-7 cells were the least sensitive to the cytotoxic activity of DMGM-12, for them IC50 = 112.08 ␮mol L−1 was almost threefold higher than that obtained for Caco-2 cells. DMGM12 showed a similar cytotoxicity towards cancer cells derived from colon (HT-29) and liver (HepG2). Addition of anionic surfactant (DBSNa) to DMGM-12 led to reduction in the cytotoxic effects of the cationic derivative towards the cell lines analysed. The exception is the MCF-7 cell line, for which the synergistic effect in cytotoxic properties was observed. As follows from the IC50 doses, the presence of DMGM-12 + DBSNa mixture suppresses the cancer cells’

Fig. 3. Effect of concentration of QDLS and their mixtures on cell viability: (a) 3.8 ␮mol L−1 , (b) 30.8 ␮mol L−1 , (c) 61.5 ␮mol L−1 , (d) 125 ␮mol L−1 ; ( ) DBSNa, and equimolar mixtures: ( ) DMGM12 + DBSNa, ( ) DMALM-12 + DBSNa, ( ) DMGM-12 + DMALM-12. DMALM-12, (

) DMGM-12, (

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)

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proliferation more effectively than individual components of this mixture. The biological activities of QDLS and their mixtures, in the concentration range from 3.8 to 125 ␮mol L−1 , were analysed. Fig. 3 shows the inhibitory effect of QDLS and their mixtures on the cancer cells. A general observation was that the compounds and mixtures tested suppressed the cancer cells’ proliferation in a dose-dependent manner. However, their cytotoxic activities significantly differed, which was particularly evident at the lowest doses. Sometimes, DMGM-12, DBSNa and DMGM-12 + DBSNa did not exhibit cytotoxic effects below the concentration of 61.5 ␮mol L−1 , as observed for the nontumorigenic colon NCM460 cell line. A strong inhibitory effect on cancer cell proliferation was observed for DMALM-12 derivative, regardless of the cell line tested. Culture supplementation with DMALM-12 or the equimolar mixtures: DMALM-12 + DBSNa, DMALM-12 + DMGM-12 in the amount of 61.5 ␮mol L−1 led to 87–99% inhibition of cancer cells’ proliferation and viability (Fig. 3c). Complete suppression of cells proliferation was noticed for DMALM-12 and their mixtures concentrations close to 125 ␮mol L−1 (Fig. 3d). On the basis of the results presented, no clear conclusion about a correlation between adsorption and biological properties could be drawn. In our previous paper [11] it has been shown that DMGM-12 + DBSNa revealed a higher surface activity than DMALM12 + DBSNa mixture, although the mixture DMALM-12 + DBSNa showed much better antitumor potential towards the cancer cell lines tested. The experiments conducted also revealed how challenging task is to find substances or mixtures selectively acting towards a specific type of cancer cells. The substance which exhibits excellent cytotoxic properties towards a given cancer cell line does not have to show similar activity towards other cancer cell lines. Each cell line tested showed different sensitivity to the mixtures and their components. Literature gives much information about the cytotoxicity of surfactants [51], their mixture [52,53] or cationic nanovesicles [26]. Thus, our observations are consistent with literature reports showing significant differences in the cytotoxic effects of chemicals towards different cell types tested [54,55]. It has been shown that the cytotoxic effects of particular carrier systems differ, depending on the cell lines used, due to the innate nature, metabolic abilities (e.g. enzymes present) and capabilities of these cells [51]. Many independent studies have shown the effectiveness of surfactants not applied alone but in combination with other penetration enhancers as co-surfactants [56]. Literature gives conflicting reports about cytotoxicity of surfactant mixtures. For the same composition of mixtures, some authors report that mixture has a higher cytotoxicity than the sum of cytotoxicties of its individual components (so-called synergistic effect in cytotoxicity), while other authors do not report it [52,57]. Moreover, Zoltán Ujhelyi et al. [52] have proved that differences in interpretation of the cytotoxicity results may be caused by various concentrations of surfactants. Buyukozturk et al. [57] indicated that cytotoxicity depends both on the type of surfactant or surfactant ratio in a mixture and their interactions. Nevertheless, surfactants in emulsion and their stable or unstable formulations can also influence the cytotoxicity. Micelles formed might affect the membrane components in different manner. All in all, the cytotoxicity mechanism may be different in non-micellar and micellar solutions [52].

4. Conclusions The results reported in this paper concern the adsorption dynamics of QDLS and its mixtures. Analysis of the dynamic surface tension isotherms has shown that the dynamics of adsorption is determined by composition of the solutions. A strong correlation

7

has been found between the surfactants concentration and the dissociation rate of micelles. The stability of micelles decreased with increasing surfactants concentration. Faster dissociation of micelles caused the formation of monomers in solution and they were able to reach the interface in a shorter time and effectively lower the surface tension. The course of dynamic surface tension curves reflects this effect. Moreover, our study has shown that the aggregates stability depends on the composition of the surfactant mixtures. The most stable aggregates are formed in equimolar mixed systems. In micellar solutions of catanionic mixtures the synergistic effect in lowering the surface tension is found. In vitro studies have indicated no direct correlation between surface activity and biological potential of individual surfactants and their mixtures. The data obtained in the cytotoxicity tests have shown that the presence of DMALM-12 in mixed systems significantly enhances the inhibition effect on proliferation of human cancer cells, regardless of the type of cell line. On the other hand, DMGM-12 shows poorer antitumor cytotoxic potential than DMALM-12, although both QDLS derivatives reveal similar surface activity. Moreover, our studies show the complexity of the mechanisms of activity of surfactants and their mixtures on the surfaces of various cell. Thus, the results obtained have highlight the difficulties in selection of specific ingredients to combat a particular type of cancer. Generally, one can conclude that cytotoxicity of surfactants and their mixtures strongly depends on the surfactants type, composition of surfactants mixture, surfactant concentration and the type of cancer cell lines.

Acknowledgment This work was supported by 32-369/14 DS-PB.

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